Abstract
BACKGROUND: Ventilator-induced diaphragmatic dysfunction (VIDD) is a major complication in critically ill patients. Prolonged mechanical ventilation (MV) triggers diaphragmatic fibrotic remodelling, but the underlying mechanisms remain unclear. This study investigated the role of the mechanosensitive channel Piezo1 in this process. METHODS: A rat model of MV was established for 6 or 12 h. Diaphragm structure (atrophy and fibrosis) and function (frequency-contraction curve and fatigue index) were assessed. The roles of Piezo1 were probed using the inhibitor GsMTx4 (a nonspecific mechanosensitive channel inhibitor) and adeno-associated virus (AAV)-mediated knockdown. Downstream signalling was identified by RNA sequencing (RNA-seq) and validated with cytosporone-B (CsnB, a specific agonist of Nr4a1). RESULTS: Compared with controls, MV for 12 h induced significant diaphragm fibrosis, atrophy and dysfunction, alongside increased Piezo1 expression (mRNA: 2.362 ± 0.429 vs. 0.920 ± 0.363, p = 0.0018; protein: 1.098 ± 0.103 vs. 0.676 ± 0.102, p = 0.0007). Both GsMTx4 and Piezo1 knockdown alleviated these effects. Knockdown reduced the collagen deposition area by approximately 21% and downregulated key fibrotic markers including fibronectin (0.749 ± 0.118 vs. 1.081 ± 0.117, p < 0.0001), collagen 1 (0.703 ± 0.087 vs. 1.155 ± 0.131, p < 0.0001), collagen 3 (0.879 ± 0.074 vs. 1.063 ± 0.068, p = 0.022) and α-SMA (0.872 ± 0.657 vs. 1.108 ± 0.078, p = 0.0031) compared to the MV12 + shCtrl group. RNA-seq identified Nr4a1 as a downstream factor (p value < 0.009). CsnB treatment increased Nr4a1 expression (1.128 ± 0.113 vs. 0.490 ± 0.084, p < 0.0001), mitigating prolonged MV-induced diaphragm fibrosis and dysfunction but not atrophy (938.1 ± 116.2 vs. 754.7 ± 155.5, p = 0.1079). CONCLUSIONS: Piezo1 upregulation is a key mechanism in ventilator-induced diaphragm fibrosis, potentially mediated through the Akt/Nr4a1 signalling pathway. Targeted inhibition of Piezo1 or activation of Nr4a1 presents a promising therapeutic strategy to prevent fibrosis and preserve diaphragm function.